The present invention provides an advanced glycation endproduct (AGE) crosslink that exhibits immunological crossreactivity with in vivo AGEs.
The glycation reaction is manifest by the appearance of brown pigments during the cooking of food, identified by Maillard in 1912. Maillard observed that glucose or other reducing sugars react with amino-containing compounds, including amino acids and peptides, to form adducts that under go a series of dehydrations and rearrangements to form stable brown pigments. Heat-treated foods undergo non-enzymatic browning as a result of a reaction between glucose and a polypeptide chain. Thus, pigments responsible for the development of brown color that develops as a result of protein glycosylation possessed characteristic spectra and fluorescent properties.
Subsequent reactions (including various dehydrations, oxidations, eliminations, condensations, cleavages and other chemical changes) occur to produce a vast array of xe2x80x9cearlyxe2x80x9d and xe2x80x9clatexe2x80x9d glycation adducts. More advanced glycation adducts are sometimes described as a class of yellow-brown, fluorescent pigments with intra- and intermolecular crosslinking activity. Specific glycation entities are thought to occur at low abundance within a widely divergent pool of advanced glycation endproducts (or AGEs). Despite significant research activity, the molecular structures of only a few of the later glycation adducts and products have been determined. Moreover, the contribution of these identified, in vivo-formed advanced glycation structures to biological processes is poorly understood. Therefore, there is a need in the art to identify AGEs and determine their biological properties.
The process of advanced glycation leads from the reversible interaction of reducing sugars with amino groups to the formation of more complex, irreversibly-bound structures with varied spectral and covalent cross-linking properties. These later products, termed advanced glycation endproducts or AGEs, form in vivo by chemical principles first described for the Maillard reaction (Ledl and Schleicher, Angew. Chem. Int. Ed. Engl. 29:565, 1990; and Maillard, C. R. Hebd. Seances Acad. Sci. 154:66, 1912). The potential significance of Maillard-type reactions in living systems however, has been appreciated only over the last 15 years and the term advanced glycation has come to refer specifically with those aspects of Maillard chemistry that involve macromolecules and which occur under physiological conditions. It is evident that AGEs form in living tissues under a variety of circumstances, and that they play an important role in protein turnover, tissue remodeling, and the pathological sequelae of diabetes and aging (Bucala and Cerami, Adv. Pharm. 23:1, 1992).
The initial event in protein glycation is the reaction of a reducing sugar such as glucose with the N-terminus of a protein or the xcex5-amnino group of a lysine to form an aldimine, or Schiff base. The Schiff base can hydrolyze back to its reactants or undergo an Amadori rearrangement to form a more stable Nxcex5-(1-deoxy-1-fructosyl) lysine (Amadori product, AP). The reaction pathway leading to reactive crosslinking moieties (i.e. AGE formation) commences by further rearrangement or degradation of the AP. Possible routes leading from AP precursors to glucose-derived, protein crosslinks has been suggested only by model studies examining the fate of the AP in vitro. One pathway proceeds by loss of the 4-hydroxyl group of the AP by dehydration to give a 1,4-dideoxy-1-alkylamino-2,3-hexodiulose (AP-dione). An AP-dione with the structure of an amino-1,4-dideoxyosone has been isolated by trapping model APs with aminoguanidine, an inhibitor of the AGE formation (Chen and Cerami, J Carbohydrate Chem. 12:731, 1993). Subsequent elimination of 5-hydroxy then gives a 1,4,5-trideoxy-1-alkylamino-2,3-hexulos-4-ene (AP-ene-dione), which has been isolated as a triacetyl derivative of its 1,2-enol form (Estendorfer et al., Angew. Chem. Ent. Ed. Engl. 29:536, 1990). Both AP-diones and AP-ene-diones would be expected to be highly reactive toward protein crosslinking reactions, for example, by serving as targets for the addition of a guanidine moiety from arginine or an xcex5-amino group from lysine.
Dicarbonyl containing compounds, such as methylglyoxal, glyoxal and deoxyglucosones, participate in condensation reactions with the side chains of arginine and lysine. For example, the addition of methylglyoxal to the guanidine moiety of arginine leads to the formation of imidazol-4-one adducts (Lo et al., J. Biol. Chem. 269:32299, 1994) and pyrimidinium adducts (Al-Abed et al., Bioorg. Med. Chem. Lett. 6:1577, 1996). In one study, Sell and Monnier isolated pentosidine, an AGE fluorescent crosslink which is a condensation product of lysine, arginine, and a reducing sugar precursor (Sell and Monnier, J. Biol. Chem. 264:21597, 1989) from human dura collagen. The mechanism of pentosidine formation remains uncertain but crosslinking requires that the lysine-bound, glucose-derived intermediate contain a dicarbonyl functionality that can react irreversibly with the guanidinium group of arginine.
Several lines of evidence have established that AGEs exist in living tissue (Bucala and Cerami, Adv. Pharm. 23:1, 1992), yet the identity of the major AGE crosslink(s) that forms in vivo remains uncertain. Recent phannacologically-based data nevertheless have affirmed the importance of the AP-dione pathway in stable crosslink formation (Vasan et al., Nature 3 82:275, 1996). The lack of precise data concerning the structure of AGEs has been attributed to the lability of AGE crosslinks to the standard hydrolysis methods employed to remove the protein backbone, and to the possible structural heterogeneity of the crosslinks themselves. Moreover, there is data to suggest that the pathologically-relevant crosslinks may not themselves be fluorescent (Dyer et al. J. Clin. Invest. 91:2463, 1993), a property that has been historically associated with AGE formation and almost universally used as an indicator of the Maillard reaction in vivo.
Hyperimmunization techniques directed against an AGE-crosslinked antigen produced both polyclonal and monoclonal antibodies that recognize in vivo formed AGEs (Makita et al., J. Biol. Chem. 267:1997, 1992). These antibodies made possible the development of immunohistochemical and ELISA-based technologies that were free of specificity and other technical problems associated with prior fluorescence-based assays, and provided the first sensitive and quantitative assessment of advanced glycation in living systems. These anti-AGE antibodies were found to recognize a class of AGEs that was prevalent in vivo but immunochemically distinct from previously characterized structures such as FFI, pentosidine, pyrraline, CML, or AFGP (Makita et al., J. Biol. Chem. 267:1992, 1992). The specific AGE epitope recognized by these antibodies increased as a consequence of diabetes or protein age on various proteins such as collagen, hemoglobin, and LDL (Makita et al., J. Biol. Chem. 267:1997, 1992; Makita et al., Science 258:651, 1992; Wolffenbuttel et al., The Lancet 347, 513, 1996; and Bucala et al. Proc. Natl. Acad. Sci. U.S.A. 91:9441, 1994). One particular polyclonal antibody species, designated xe2x80x9cRUxe2x80x9d, has been employed in an ELISA assay tested in human clinical trials. Immunoreactive AGEs were found to be inhibited from forming by administration of the pharmacological inhibitor, aminoguanidine (Makita et al., Science 258:651, 1992; and Bucala et al. Proc. Natl. Acad. Sci. U.S.A. 91:9441, 1994), and to provide important prognostic information correlated to diabetic renal disease (Beisswenger et al. Diabetes 44:824, 1995).
Despite the increasing body of data implicating the advanced glycation pathway in the etiology of such age- and diabetes-related conditions as atherosclerosis, renal insufficiency, and amyloid deposition, elucidation of the structure(s) of the pathologically important, AGE-crosslinks that form in vivo has been a challenging problem. Investigations of AGEs that form in vivo have necessarily relied on chemical methods to purify the crosslinking moieties away from their macromolecular backbones. These studies have led to a recognition that the major crosslinks which form in vivo are largely acid-labile and non-fluorescent (Bucala and Cerami, Adv. Pharm., 23:1, 1992; Sell and Monnier, J. Biol. Chem. 264:21597,1989; and Dyer et al. J. Clin. Invest. 91:2463, 1993). However, in view of a predictive antibody-based (ELISA) diagnostic assay, there is a need in the art to isolate and identify immunogenic AGEs that can be used to both standardize and improve such diagnostic assays. The present invention was made in an effort to achieve the foregoing goals. Further, there is a need in the art to measure formation of advanced glycosylation endproducts in all applications where protein aging is a serious detriment. This includes, for example, the area of food technology (ie., determination of the amount of food spoilage), perishability or shelf-life determination of proteins and other amino-containing biomolecules.
The present invention provides a means for standardizing a kit that provides a means for measuring the formation of AGEs as a diagnostic assay. The present invention further provides a novel isolate AGE that is antigenic and useful for forming antibodies having utility in diagnostic assays and for standardizing diagnostic assays.
The invention provides a condensation product advanced glycation endproduct (AGE) comprising a lysine component, an arginine component and a reducing sugar component. Preferably, the condensation product is an AGE according to formula I: 
wherein the lysine component is indicated by the box labeled xe2x80x9cKxe2x80x9d; the arginine component is indicated by the box labeled xe2x80x9cRxe2x80x9d; and the reducing sugar component is not boxed; and wherein R1 and R4 are independently H or an amide bond to an amino acid residue or a peptide chain; R2 and R3 are, independently, OH or an amide bond to an amino acid residue or a peptide chain; R5 is H, CH2OH or CHOHCH2OH; and wherein if more than one of R1, R2, R3 or R4 is an amide bond, then the lysine xe2x80x9cKxe2x80x9d component and the arginine xe2x80x9cRxe2x80x9d component may be amino acid residues of the same or a different peptide chain. Most preferably, the condensation product is an ALI having the structure: 
wherein Z is H, carboxybenzoyl, or the remainder of the polypeptide linked to the Arg and Lys groups; and Y is OH or the remainder of the polypeptide.
The present invention further provides a method for increasing macrophage recognition and elimination of advanced glycosylation endproducts, comprising administering to a mammal a therapeutic amount of a compound of formula I.